sj-docx-1-nms-10.1177_14614448221128475 – Supplemental material for The online structure and development of posting behaviour in Dutch anti-vaccination groups on Telegram

Supplemental material, sj-docx-1-nms-10.1177_14614448221128475 for The online structure and development of posting behaviour in Dutch anti-vaccination groups on Telegram by Anniek Schlette, Jan-Willem van Prooijen, Arjan Blokland and Fabienne Thijs in New Media & Society</p

Housing conditions.

<p>Pictures of the 3 different housing conditions; solitary housed animals (A), socially housed animals (B), and the animals housed in an enriched environment (C). Of note, the enriched environment picture only represents the object arrangement during one particular week, the constellation and number of items was changed weekly.</p

ORT 48 h intervals.

<p>Discrimination performance (d2, mean + SEM) of SOL (A), SOC (B) and EE (C) rats after administration of different doses of vardenafil in the object recognition task. All housing conditions were tested in a 48 h retention interval in combination with 0, 0.1, 0.3 and 1 mg/kg vardenafil (x-axis), EE animals also received 0.03 mg/kg vardenafil. A difference from zero is indicated with hash symbols (one-sample <i>t</i>-test; ###: <i>p</i><0.001), differences from the vehicle condition (0 mg/kg) within the same retention interval are indicated with asterisks (Tukey HSD; **: <i>p</i><0.01; ***: p<0.001). Of note, the 0 mg/kg vardenafil conditions are identical to those used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111692#pone-0111692-g002" target="_blank">Figure 2</a>.</p

PDE5 Inhibition Improves Object Memory in Standard Housed Rats but Not in Rats Housed in an Enriched Environment: Implications for Memory Models?

<div><p>Drug effects are usually evaluated in animals housed under maximally standardized conditions. However, it is assumed that an enriched environment (EE) more closely resembles human conditions as compared to maximally standardized laboratory conditions. In the present study, we examined the acute cognition enhancing effects of vardenafil, a PDE5 inhibitor, which stimulates protein kinase G/CREB signaling in cells, in three different groups of male Wistar rats tested in an object recognition task (ORT). Rats were either housed solitarily (SOL) or socially (SOC) under standard conditions, or socially in an EE. Although EE animals remembered object information longer in the vehicle condition, vardenafil only improved object memory in SOL and SOC animals. While EE animals had a heavier dorsal hippocampus, we found no differences between experimental groups in total cell numbers in the dentate gyrus, CA2–3 or CA1. Neither were there any differences in markers for pre- and postsynaptic density. No changes in PDE5 mRNA- and protein expression levels were observed. Basal pCREB levels were increased in EE rats only, whereas β-catenin was not affected, suggesting specific activation of the MAP kinase signaling pathway and not the AKT pathway. A possible explanation for the inefficacy of vardenafil could be that CREB signaling is already optimally stimulated in the hippocampus of EE rats. Since previous data has shown that acute PDE5 inhibition does not improve memory performance in humans, the use of EE animals could be considered as a more valid model for testing cognition enhancing drugs.</p></div

ORT 24 h intervals.

<p>Discrimination performance (d2, mean + SEM) of SOL (A), SOC (B) and EE (C) rats after administration of different doses of vardenafil in the object recognition task. All housing conditions were tested in a 24 h retention interval in combination with 0, 0.1, 0.3 and 1 mg/kg vardenafil (x-axis). A difference from zero is indicated with hash symbols (one-sample <i>t</i>-test; #: <i>p</i><0.05; ##: <i>p</i><0.01; ###: <i>p</i><0.001), differences from the vehicle condition (0 mg/kg) within the same retention interval are indicated with an asterisk (Tukey HSD; *: <i>p</i><0.05). Of note, the 0 mg/kg vardenafil conditions are identical to those used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111692#pone-0111692-g002" target="_blank">Figure 2</a>.</p

Results stereological and biochemical measurements.

<p>Comparisons of the stereological and biochemical measurements between the different housing conditions using one-way ANOVA and Tukey HSD post hoc analysis. Individual cells show the mean (+SEM) of each housing condition. Significant differences between SOL and EE animals are indicated by asterisks (⁽*⁾: P = 0.052; *: p<0.05; ***: p<0.001), significant differences between SOL and SOC animals are indicated by hash signs (##: p<0.01) and significant differences between SOC and EE animals are indicated by plus signs (+: p<0.05; ++: p<0.01).</p><p>Results stereological and biochemical measurements.</p

Exploration levels in the ORT.

<p>This table shows the number of animals (n) and mean exploration time (± SEM) in seconds on both the objects in T1 (e1) and T2 (e2) in every treatment condition. SOL, SOC, and EE animals were tested repeatedly, so every individual animal performed each experimental condition once. The 1 h retention interval was tested first, followed by the 24 h interval conditions. Finally, the 48 h retention interval conditions were tested. Of note, although a total number of 22 SOL, 18 SOC and 18 EE animals were tested in each condition, animals that explored less than 6 s in T1 and/or less than 9 s in T2 were excluded from the analyses.</p><p>Exploration levels in the ORT.</p

Effect of retention delay on object discrimination.

<p>Discrimination performance (d2, mean + SEM) of SOL, SOC and EE rats treated with vehicle in a 1 h, and 24 h and 48 h retention interval (x-axis) in the object recognition task. A difference from zero is indicated with hash symbols (one-sample <i>t</i>-test; #: <i>p</i><0.05; ###: <i>p</i><0.001), differences between conditions within the same retention interval are indicated with asterisks (Tukey HSD; (*): p<0.10; *: <i>p</i><0.05).</p

Effects of corticosterone (CORT) administration via the drinking water upon water intake (mean % change+S.E.M.) during the 12 week treatment period.

<p>Significant increased amounts of water intake in the CORT group are denoted with *(<i>P</i><0.01).</p

Plasma glucose levels (mean % change from baseline+S.E.M.) after acute glucose challenge in fasted animals receiving corticosterone (CORT) or vehicle either orally via the drinking water or by daily s.c. injection as measured by oral glucose tolerance tests (OGGT).

<p>Within each OGTT, plasma glucose levels are measured at baseline (0) and 15, 30, 60 and 120 min after an oral administration of glucose. Treatment effects upon glucose tolerance were measured at 3 time-points throughout the experiment, namely directly after 4 weeks treatment (OGTT1) and 1 and 2 weeks after treatment cessation (OGTT2, OGTT3, respectively). (*) <i>trend</i>; <i>P</i> = 0.094.</p